Structural and regulatory studies on protein components of the Escherichia coli (E. coli) sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate on an active site histidine and that phosphoryl group can be transferred to the active site of the second component (HPr). EI contains two domains. The isolated recombinant helical domain was shown to bind to HPr. In vitro reconstitution experiments with isolated recombinant helical domain and a construct deficient in that domain showed that it was possible to recover autophosphorylation by PEP. Phosphotransfer activity was also recovered in reconstitution of the aminoterminal and carboxyterminal domains. Mutations at the active site of EI were engineered and studied by biophysical methods for determination of conformational stability. The order of conformational stability is His>Ala>Glu>His-PO3. The binding of HPr to active-site mutants is temperature-independent with about the same affinity constant. One of the PTS proteins that can accept a phosphoryl group from P-HPr is named IIAglc. This protein also plays a role in the regulation of activity of other sugar transport systems in E. coli. By using a previously described direct binding assay, a region on the surface of IIAglc that interfaces with lactose permease was characterized. Actetylation of lysine residues by sulfosuccinimidyl acetate treatment of IIAglc reduced the degree of interaction with lactose permease. To localize the lysine residues on IIAglc that are involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were able to bind to lactose permease. A model for the region of the surface of IIAglc, including Lys69, that interacts with lactose permease was proposed. HPr is a multifunctional protein; as a phosphocarrier in the PTS, it interacts with both EI and IIAglc. In addition it is an allosteric regulator of glycogen phosphorylase. Because the nature of the surface of HPr that interacts with this multiplicity of proteins was previously undefined, those interactions were investigated by NMR. The chemical shift changes of the backbone and side chain amide 1H and 15N nuclei of uniformly 15N-labeled HPr in the presence and absence of natural abundance glycogen phosphorylase, IIAglc or the aminoterminal domain of EI were determined. Mapping those chemical shift perturbations on the three-dimensional structure of HPr permitted the identification of the binding surface on HPr for interaction with those proteins. The mapped interfaces are remarkably similar, indicating that HPr employs a similar syurface in binding to its partners.